Methods and Materials for Expression of a Recombinant Protein

ABSTRACT

Recombinant expression vectors are provided comprising a 3′ UTR of a light chain and an Epstein-Barr virus origin of replication. Also provided are host cells comprising such vectors and methods of producing recombinant protein with such vectors. Additional methods of producing a recombinant protein involve contacting cells with a first and second vector, each of which encode a different polypeptide chain, and wherein the second vector is present in an amount which is about 1.5 to 2.5 times as much as that of the first vector. Cells also can be transfected with a recombinant transient expression vector encoding a protein and are cultured in a medium in a membrane-enhanced culturing vessel to produce recombinant protein.

FIELD OF THE INVENTION

This invention pertains to methods of producing a recombinant proteinand recombinant expression vectors and host cells for use therein.

BACKGROUND OF THE INVENTION

Large-scale transient expression of recombinant proteins has been anarea of rapid development in the past several years as an alternative orprecursor to stable cell line development to generate multi-milligramquantities of protein (Wurm et al., Curr. Opn. Biotech. 10: 156-159(1999)). Human embryonic kidney (HEK293) cells are one of the mostwidely used cell lines for transient expression and have beensuccessfully adapted to suspension-growth to help facilitate culturescale-up. Recent reports have successfully demonstrated the usage oftransiently expressing suspension-adapted HEK293 cells in 1-3 L culturesto generate recombinant proteins including soluble polypeptides,transmembrane proteins, and human antibodies (Durocher et al., NucleicAcids Res. 30:1-9 (2002); Meissner et al., Biotechnol. Bioeng. 75:197-203 (2000); and Cote et al., Biotechnol. Bioeng. 59: 567-575(1998)).

In particular, Durocher et al. has shown that HEK293E cells expressingthe Epstein-Barr virus (EBV) nuclear antigen-1 protein (EBNA1) were ableto routinely generate >10 mg/L of a number of different recombinantproteins using the cationic polymer transfection reagent,polyethyleneimine (PEI) (Boussif et al., Proc. Natl. Acad. Sci. 92:7297-7301 (1995); and Mislick et al., Proc. Natl. Acad. Sci. 93:12349-12354 (1996)).

Despite these advances, there is still a need in the art for improvedexpression systems including optimized transient transfection systemsfor time- and cost-efficient production of recombinant proteins. Theinvention provides such optimized methods of producing recombinantproteins. These and other advantages of the invention, as well asadditional inventive features, will be apparent from the description ofthe invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides recombinant expression vectors useful in methodsof producing a recombinant protein. One of the inventive recombinantexpression vectors comprises a 3′ untranslated region (UTR) of a lightchain gene. Another recombinant expression vector provided hereincomprises a 3′ UTR and an Epstein-Barr virus origin of replication. Hostcells comprising any of the inventive recombinant expression vectors arealso provided herein.

The invention further provides methods of producing a recombinantprotein. In a first method, the recombinant protein is a heterodimericor heteromultimeric protein, either of which comprises a firstpolypeptide chain and a second polypeptide chain, wherein the firstpolypeptide chain is different from the second polypeptide chain. Themethod comprises contacting cells in a medium with a first vector and asecond vector, wherein the first vector encodes the first polypeptidechain and the second vector encodes the second polypeptide chain, andthe second vector is present in the medium in an amount which is about1.5 to about 2.5 times as much as the amount of the first vector.

In a second method of producing a recombinant protein, the methodcomprises culturing cells, which have been contacted with a recombinanttransient expression vector encoding the protein, in a medium in amembrane-enhanced culturing vessel, whereupon a recombinant protein isproduced. Alternatively, the second method comprises culturing cells,which have been contacted with a recombinant transient expression vectorencoding the protein, in a medium in a Fernbach flask.

In a third method, the recombinant protein is produced upon contactingcells with at least one of the inventive recombinant expression vectorsdescribed herein. In a fourth method, the recombinant protein isproduced upon culturing host cells comprising any of the inventiverecombinant expression vectors described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an illustration of the pMXT recombinant expression vectorwithout any recombinant protein coding sequences. FIG. 1B is anillustration of the pMXT vector encoding a human γ₂ heavy chain, whileFIG. 1C is an illustration of the pMXT vector encoding a human κ lightchain. The following abbreviations are used in FIGS. 1A-1C: Ap,ampicillin resistance marker; CMV promoter, cytomegalovirus promoter;MCS, multiple cloning sequence; 5′ UT intron, 5′ untranslated regionintron; SP, signal peptide; V, variable region; C, constant region; LC3′ UT, light chain 3′ untranslated region, OriP, Epstein-Barr virusorigin of replication; pUC19ori, origin of replication from the pUC19plasmid.

FIGS. 2A-2I are flow cytometry data graphs depicting the levels offluorescence of green fluorescence protein (GFP) and propidium iodide(PI) under differing transfection conditions, specifically differing DNAand polyethyleneimine (PEI) concentrations. In FIG. 2A, cells weretransfected with 1 μg/ml DNA and 1 μg/ml PEI. In FIG. 2B, cells weretransfected with 2 μg/ml DNA and 2 μg/ml PEI. In FIG. 2C, cells weretransfected with 5 μg/ml DNA and 5 μg/ml PEI. In FIG. 2D, cells weretransfected with 1 μg/ml DNA and 2 μg/ml PEI. In FIG. 2E, cells weretransfected with 2 μg/ml DNA and 4 μg/ml PEI. In FIG. 2F, cells weretransfected with 5 μg/ml DNA and 10 μg/ml PEI. In FIG. 2G, cells weretransfected with 1 μg/ml DNA and 5 μg/ml PEI. In FIG. 2H, cells weretransfected with 2 μg/ml DNA and 10 μg/ml PEI. In FIG. 2I, cells weretransfected with 5 μg/ml DNA and 25 μg/ml PEI.

FIG. 3 is a graph showing the % cell viability (X) and % GFP positive293E cells (▪) that were adapted to suspension growth in differentserum-free media and optimized for transfection. A control set of 293Ecells were grown in DMEM.

FIG. 4 is a graph showing the antibody production by cells which wereco-transfected with different heavy chain (HC):light chain (LC) ratiosof different vector types.

FIG. 5A is a graph showing antibody production (•) and cell viability(X) of transiently transfected 293E cells in Integra flasks as afunction of time post-transfection.

FIG. 5B is a graph showing the production of antibodies in shake flasksvs. Integra flasks by transfected cell cultures at day 7post-transfection.

FIG. 6A is a graph of the percentage of viable cells transfected withDNA encoding Ab#1 as a function of time post-transfection. FIG. 6B is agraph of the percentage of viable cells transfected with DNA encodingAb#2 as a function of time post-transfection. In both FIGS. 6A and 6B, ▪is I-50; ▴ is 1-100; • is I-200; ♦ is I-400; and X is E-200.

FIG. 7A is a graph of the number of viable cells transfected with DNAencoding Ab#1 as a function of time post-transfection. FIG. 7B is agraph of the number of viable cells transfected with DNA encoding Ab#2as a function of time post-transfection. In both FIGS. 7A and 7B, ▪ isI-50; ▴ is 1-100; • is I-200; ♦ is I-400; and X is E-200.

FIG. 8A is a graph of the concentration of antibody produced by cellstransfected with DNA encoding Ab#1 as a function of time posttransfection. FIG. 8B is a graph of the concentration of antibodyproduced by cells transfected with DNA encoding Ab#2 as a function oftime post-transfection. In both FIGS. 8A and 8B, ▪ is I-50; ▴ is 1-100;• is 1-200; ♦ is I-400; and X is E-200.

FIG. 9A is a graph of the total antibody produced by cells tranfectedwith DNA encoding Ab#1 as a function of time post-transfection. FIG. 9Bis a graph of the total yield of antibody produced by cells transfectedwith DNA encoding Ab#2 as a function of time post-transfection.

FIG. 10 shows SEQ ID NO: 1, which is the nucleotide sequence of pMXT5(FIG. 1A) without any coding sequences. Restriction enzyme sites arelabeled with the name of the enzyme above the position of the site. CMVpromoter comprises nucleotides 1-1037; 5′ UTR intron comprisesnucleotides 888-974; MCS comprises nucleotides 1038-1061; LC 3′ UTcomprises nucleotides 1062-2560; OriP comprises nucleotides 2561-4550;pUC19 ori comprises nucleotides 4551-5220; and Ap comprises nucleotides5221-6380.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides recombinant expression vectors useful in methodsof producing a recombinant protein. One of the inventive recombinantexpression vectors comprises a 3′ untranslated region (UTR) of a lightchain gene. Another recombinant expression vector provided hereincomprises a 3′ UTR and an Epstein-Barr virus origin of replication(oriP). Inventive recombinant expression vectors optionally comprise apUC19 origin of replication (pUC19ori).

For purposes herein, the term “recombinant expression vector” means agenetically-modified oligonucleotide (i.e., polynucleotide) constructthat permits the production of a protein within a cell, when theconstruct comprises a nucleotide sequence encoding the protein, and theconstruct is contacted with the cell under conditions sufficient to havethe protein expressed within the cell. As the expression vector isrecombinant, the vector of the invention is not naturally-occurring as awhole. However, parts of the vectors can be naturally-occurring.

The recombinant expression vector can comprise any type of nucleotides,including, but not limited to DNA and RNA, which can be single-strandedor double-stranded, which can be synthesized or obtained in part fromnatural sources, and which can contain natural or non-natural or alterednucleotides. Examples of non-natural or altered nucleotides that can beused to generate the recombinant expression vectors include, but are notlimited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3) w, and 2,6-diaminopurine.

The recombinant expression vector can comprise naturally-occurring ornon-naturally-occurring internucleotide linkages, or both types oflinkages, such as phosphoroamidate linkages or phosphorothioatelinkages, instead of the phosphodiester linkages found between thenucleotides of an unmodified oligonucleotide. Preferably, thenon-naturally occurring or altered nucleotides or internucleotidelinkages do not hinder in any way the transcription or replication ofthe vector.

The recombinant expression vector can be any suitable recombinantexpression vector, and can be used to transform or transfect anysuitable host. For example, one of ordinary skill in the art appreciatesthat transformation or transfection is a process by which, for example,exogenous nucleic acids such as DNA are introduced into cells whereinthe transformation or transfection process involves contacting the cellswith the exogenous nucleic acids such as the recombinant expressionvector as described herein. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used.Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3,pBI121, and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-Cl, pMAM, and pMAMneo (Clontech). A preferred recombinantexpression vector includes the pMXT vector as shown in FIGS. 1A-1C.

The recombinant expression vector can be prepared using standardrecombinant DNA techniques described in, for example, Sambrook et al.,Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, New York, N.Y. (1994).

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

A construct of a recombinant expression vector, which is circular orlinear, can be prepared to contain a replication system functional in aprokaryotic or eukaryotic host cell. Replication systems can be derivedfrom ColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, and the like.The recombinant expression vector of the invention can comprise areplication system, which comprises an oriP. Preferably, the inventiverecombinant expression vector comprises an oriP, and not an Epstein Barrvirus nuclear antigen (EBVNA), which EBVNA is known to activate an oriP.

As used herein, the term “oriP” or “Epstein-Barr virus origin ofreplication” refers to a nucleotide sequence that is substantiallyidentical to the Epstein-Barr virus origin of replication, which has thenucleotide sequence of nucleotides 2561-4550 of SEQ ID NO: 1. It ispreferred that no insertions, deletions, inversion, and/or substitutionsare present in this nucleotide sequence. However, one of ordinary skillin the art appreciates that the nucleotide sequence of nucleotides2561-4550 of SEQ ID NO: 1 can have insertions, deletions, inversion,and/or substitutions that will not negatively affect the function of thenucleotide sequence, which is to promote high copy episomal plasmidreplication. One of ordinary skill in the art further appreciates thatsuch high copy episomal plasmid replication occurs in mammalian cells.

The recombinant expression vector also preferably comprises a pUC19origin of replication. As used herein, the term “pUC19 origin ofreplication” refers to the nucleotide sequence of the origin ofreplication from a pUC19 vector, which is commercially available fromFermentas Life Sciences and has the nucleotide sequence of nucleotides4551-5220 of SEQ ID NO: 1. It is preferred that no insertions,deletions, inversion, and/or substitutions are present in thisnucleotide sequence. However, one of ordinary skill in the artappreciates that nucleotides 4551-5220 of SEQ ID NO: 1 can haveinsertions, deletions, inversion, and/or substitutions that will notaffect the function of the nucleotide sequence, which is to promote highcopy episomal plasmid replication. One of ordinary skill in the artfurther appreciates that such high copy episomal plasmid replicationoccurs in bacterial cells.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the inventiverecombinant expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or normativepromoter operably linked to the nucleic acid encoding the protein. Theselection of promoters, e.g., strong, weak, inducible, tissue-specific,and developmental-specific, is within the ordinary skill in the art.Similarly, the combining of a nucleic acid with a promoter is alsowithin the skill in the art. The promoter can be a viral promoter or anon-viral promoter. Preferably, the promoter is a viral promoter. Morepreferably, the viral promoter is a strong viral promoter, such as acytomegalovirus (CMV) promoter. The CMV promoter is known in the art andhas the nucleotide sequence of nucleotides 1-1037 of SEQ ID NO: 1. It ispreferred that no insertions, deletions, inversion, and/or substitutionsare present in this nucleotide sequence. However, one of ordinary skillin the art appreciates that nucleotides 1-1037 of SEQ ID NO: 1 can haveinsertions, deletions, inversion, and/or substitutions that will notaffect the function of the nucleotide sequence, which is to drive thetranscription of the recombinant protein coding sequence.

The recombinant expression vector comprises a 3′ UTR of a light chaingene. Preferably, the recombinant expression vector comprises a 3′ UTRof a light chain gene in combination with an Epstein-Barr virus originof replication (oriP). As used herein, the term “3′ UTR” refers to anucleotide sequence of a gene that is untranslated and is located 3′ tothe stop codon of the coding sequence of that gene. The phrase “lightchain gene” refers to a gene encoding a light chain of animmunoglobulin. Thus, in regard to the invention, the 3′ UTR of a lightchain gene is a nucleotide sequence that is originally found in alight-chain gene and that is inserted into the inventive vector. Thelight chain gene can be a light chain gene of any mammal, such as ahuman, mouse, rat, goat, rabbit, horse, pig, etc. Preferably, the lightchain gene is a mouse (murine) light chain gene. More preferably, themouse light chain gene has the nucleotide sequence of nucleotides1062-2560 of SEQ ID NO: 1. It is preferred that no insertions,deletions, inversion, and/or substitutions are present in thisnucleotide sequence. However, one of ordinary skill in the artappreciates that nucleotides 1062-2560 of SEQ ID NO: 1 can haveinsertions, deletions, inversion, and/or substitutions that will notaffect the function of the nucleotide sequence, which is to providesignals for polyadenylation. With respect to the inventive vectors, the3′ UTR of a light chain gene is preferably located immediately 3′ to thestop codon of the coding sequence of the vector. If no coding sequenceis present, then the 3′ UTR of a light chain gene preferably is located3′ to the multiple cloning sequence and/or the CMV promoter. Therecombinant expression vector can comprise a single copy of a 3′ UTR ormultiple copies of a 3′ UTR. Preferably, the recombinant expressionvector comprises a single copy of a 3′ UTR.

The recombinant expression vector preferably comprises a 5′ UTR intron.As used herein, the term “5′ UTR intron” refers to a nucleotide sequencethat is transcribed but is removed by RNA splicing and thus not retainedin the final transcript. It further is not translated and, thus, is notexpressed as part of the protein, polypeptide, or peptide encoded by thevector. The 5′ UTR intron is preferably located after the promoter inthe 5′ untranslated region of the recombinant expression vector. The 5′UTR intron promotes enhanced expression. The 5′ UTR intron can be fromany naturally-occurring source or can be constructed from portions ofdifferent sources, e.g., constructed from splice donor and acceptorsequences from different sources. For example, the 5′ UTR introncomprises a portion of a CMV intron and a portion of a SV40 16S intron.Preferably, the splice donor for the 5′ UTR intron is from the sequencedownstream of the start of transcription from the viral promoter, andthe splice acceptor is from the SV40 16S intron and has the nucleotidesequence of nucleotides 888-974 of SEQ ID NO: 1. It is preferred that noinsertions, deletions, inversion, and/or substitutions are present inthis nucleotide sequence. However, one of ordinary skill in the artappreciates that nucleotides 888-974 of SEQ ID NO: 1 can haveinsertions, deletions, inversion, and/or substitutions that will notaffect the function of the nucleotide sequence, which is to drive thetranscription of the recombinant protein coding sequence.

In a preferred embodiment, the recombinant expression vector comprises a3′ UTR, an oriP, a pUC19 origin of replication, a viral promoter, and a5′ UTR intron. Preferably, the viral promoter is a CMV promoter and the5′ UTR intron comprises a portion of a CMV intron and a portion of aSV40 16S intron, e.g., comprises nucleotides 888-974 of SEQ ID NO: 1.Most preferably, the recombinant expression vector is the vector plasmidpMXT5, which is shown pictorially in FIG. 1A (pMXT5), and which has thenucleotide sequence (without any coding sequences) of SEQ ID NO: 1 (FIG.10). For example, as shown in FIG. 10, the CMV promoter comprisesnucleotides 1-1037; 5′ UTR intron comprises nucleotides 888-974; MCScomprises nucleotides 1038-1061; LC 3′ UT comprises nucleotides1062-2560; OriP comprises nucleotides 2561-4550; pUC19 ori comprisesnucleotides 4551-5220; and Ap comprises nucleotides 5221-6380.

The recombinant expression vector can be designed for either transientexpression or for stable expression. Preferably, the vector of theinvention promotes transient expression, i.e., is a recombinanttransient expression vector, such that the vector is one that does notintegrate into the genome of a host cell. Without being bound to anyparticular theory, it is believed that the recombinant expression vectorcan be made to be a transient expression vector by incorporating intothe vector an oriP, which promotes high copy episomal plasmidreplication.

The recombinant expression vector can comprise a nucleic acid sequenceencoding any protein, such as a hormone, growth factor, antibody,receptor, structural protein, enzyme, etc. The protein can be, forexample, a therapeutic protein, and can be naturally-occurring ornon-naturally occurring, e.g., a genetically engineered proteinincluding, for example, a fusion protein, a chimeric protein, etc.Preferably, the recombinant expression vector comprises such a nucleicacid for the expression of the protein. It is to be understood that theterm “protein” as used herein includes parts or fragments thereof, andthus, polypeptides and peptides of any length are included within themeaning of this term. For example, polypeptides and peptides areincluded wherein the polypeptides can comprise, for instance, about 50or more amino acids and the peptides can comprise, for instance, about8-49 amino acids. The nucleic acid sequence encoding the protein can beobtained from any source, e.g., isolated from nature, syntheticallygenerated, isolated from a genetically-engineered organism, and thelike. An ordinarily skilled artisan will appreciate that any type ofnucleic acid sequence (e.g., DNA, RNA, genomic DNA, and cDNA) that canbe inserted into a recombinant expression vector can be used inconnection with the invention. For example, the nucleic acid sequenceencoding a protein can be naturally-occurring, e.g., a gene.Alternatively, the nucleic acid sequence encoding a protein can benon-naturally occurring, e.g., non-native to any organism, e.g., mammal.For instance, the nucleic acid sequence can be a codon optimized nucleicacid sequence in which codons within the nucleic acid sequence, whichcodons are not generally used by the host cell translation system,termed “rare codons,” are changed by in vitro mutagenesis to preferredcodons without changing the amino acids of the synthesized protein(Bradel-Tretheway et al., J. Virol. Meth., 111: 145-156 (2003);Ramakrishna et al., J. Virol. 78: 9174-9189 (2004)). In addition, thenucleic acid sequence encoding a protein can be further modified, e.g.,codon optimized, to improve the folding of the RNA, such that thefolding of the RNA transcript encoded by the nucleic acid sequence isminimized. Whatever type of nucleic acid sequence is used, the nucleicacid sequence preferably encodes a secreted protein. By “secreted” ismeant that the protein is released from the cell into the extracellularenvironment, thereby facilitating the purification of the protein. Inthis regard, the recombinant expression vector preferably comprises asignal sequence, which causes the expressed protein to be secreted fromthe cell by which it was expressed.

In a preferred embodiment, the recombinant expression vector comprises anucleic acid encoding an immunogloblin chain, e.g., light chain or heavychain. The immunoglobulin chain can be any immunoglobulin chain derivedfrom any source, genetically-modified, or synthesized. Preferably, theimmunoglobulin chain is a human immunoglobulin chain selected from thegroup consisting of a γ₁ heavy chain, a γ₂ heavy chain, a γ₄ heavychain, a κ light chain, and a λ light chain. Exemplary heavy chainconstant region sequences include: a γ₁ heavy chain constant region,which is encoded by the nucleotide sequence of SEQ ID NO: 4 andcomprises the amino acid sequence of SEQ ID NO: 5; a γ₂ heavy chainconstant region, which is encoded by the nucleotide sequence of SEQ IDNO: 6 and comprises the amino acid sequence of SEQ ID NO: 7; and a γ₄heavy chain constant region, which is encoded by the nucleotide sequenceof SEQ ID NO: 8 and comprises the amino acid sequence of SEQ ID NO: 9.Exemplary light chain constant region sequences include: a κ light chainconstant region, which is encoded by the nucleotide sequence of SEQ IDNO: 10 and comprises the amino acid sequence of SEQ ID NO: 11, and a λlight chain constant region, which is encoded by the nucleotide sequenceof SEQ ID NO: 12 and comprises the amino acid sequence of SEQ ID NO: 13.Exemplary antibody heavy and light chains include: an LDP-01 heavychain, which is encoded by the nucleotide sequence of SEQ ID NO: 14 andcomprises the amino acid sequence of SEQ ID NO: 15, and an LDP-01 lightchain, which is encoded by the nucleotide sequence of SEQ ID NO: 16 andcomprises the amino acid sequence of SEQ ID NO: 17. The LDP-01 antibodyis referred to herein as Ab#1 and has been described in WO 2004/033693(PCT/US2003/010154) and U.S. Patent Application Publication No.2003/0203447 A1.

In this regard, the recombinant expression vector desirably comprises anantibody signal sequence, which promotes the secretion of the antibodyinto the extracellular environment. Suitable antibody signal sequencesare known in the art. For example, a preferred signal sequence comprisesSEQ ID NO: 2 or SEQ ID NO: 3.

The recombinant expression vector can alternatively comprise a nucleicacid sequence encoding a functional fragment of a protein. The term“functional fragment” which is synonymous with “functional part” or“functional portion,” when used in reference to a protein, refers to anypart or fragment of the protein, which part or fragment retains abiological activity of the protein of which it is a part. Functionalfragments encompass, for example, those parts of a protein (the parentprotein) that retain a function of the parent protein to a similarextent, the same extent, or to a higher extent, as the parent protein.For instance, if the protein is an immunoglobulin, functional fragmentsthereof can include any portion of the immunoglobulin which, forexample, retains the ability to bind to the antigen of the parentimmunoglobulin. Also, for example, if the protein is a cell surfacereceptor, functional fragments thereof can include any portion of thecell surface receptor which, for instance, retains the ability to bindto the ligand of the parent cell surface receptor. In reference to theparent protein, the functional fragment can comprise, for instance,about 10%, 25%, 30%, 50%, 60%, 80%, 90%, 95%, or more of the parentprotein. The functional portion can comprise additional amino acids atthe amino or carboxy terminus of the portion, or at both termini, whichadditional amino acids are not found in the amino acid sequence of theparent protein. Desirably, the additional amino acids do not interferewith the biological function of the functional portion.

The invention further provides a host cell comprising any of therecombinant expression vectors described herein. As used herein, theterm “host cell” refers to any type of cell that can contain theinventive recombinant expression vector. The host cell can be aeukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The cell can be a culturedcell or a primary call, i.e., isolated directly from an organism, e.g.,a human. The cell can be an adherent cell or a suspended cell, i.e., acell that grows in suspension. Suitable host cells are known in the artand include, for instance, DH5α E. coli cells, Chinese hamster ovarian(CHO) cells, monkey VERO cells, COS cells, HEK293 cells, and the like.For purposes of amplifying or replicating the recombinant expressionvector, the host cell is preferably a prokaryotic cell. More preferably,the host cell is a DH5α cell. For purposes of producing a recombinantprotein, the host cell is preferably a mammalian cell. Most preferably,the host cell is a human cell. While the cell can be any cell of thehuman body, it is preferred that the cell is a human embryonic kidneycell. More preferred is that the human embryonic kidney cell expressesan Epstein Barr virus nuclear antigen-1 (EBNA-1) protein, e.g., a 293Ecell.

As used herein, the term “mammal” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Camivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

The invention further provides methods of producing a recombinantprotein. In a first method, the recombinant protein is a heterodimericor heteromultimeric protein comprising a first polypeptide chain and asecond polypeptide chain, wherein the first polypeptide chain isdifferent from the second polypeptide chain. The first method comprisescontacting cells in a medium with a first vector and a second vector,wherein the first vector encodes the first polypeptide chain and thesecond vector encodes the second polypeptide chain, and the secondvector is present in the medium in an amount which is about 1.5 to about2.5 times as much as the amount of the first vector, whereupon arecombinant protein is produced. The first and second vectors can be anysuitable vector and preferably are inventive recombinant expressionvectors as described herein.

For purposes of the first inventive method of producing a protein, thefirst and second vectors can independently be any type of vector, i.e.,the first and second vectors can have the same regulatory elements butdiffer only in the recombinant protein coding sequence containedtherein. By way of example, both the first vector and second vector canbe the pMXT vector as shown in FIG. 1A. Preferably, each of the firstvector and the second vector is one of the inventive recombinantexpression vectors described herein. Most preferably, the first andsecond vectors are pMXT vectors. For example, it is preferred that eachof the first and the second vector is a recombinant transient expressionvector. It is also preferred that each of the first and second vectorcomprises a 3′ UTR of a light chain gene and an oriP. It is alsopreferred that each of the first and second vector comprises a viralpromoter, a pUC19 origin of replication, a 5′ UTR intron, or acombination of any of the foregoing. Preferably, the viral promoter is aCMV promoter, and the 5′ UTR intron comprises nucleotides 888-974 of SEQID NO: 1. Moreover, it is preferred that each of the first and secondvector comprises an antibody signal sequence.

Also, with respect to the first inventive method of producing a protein,the second vector is present in the medium in an amount which is about1.5 to about 2.5, e.g., 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.125, 2.25, 2.3,2.4, and 2.5, times as much as the amount of the first vector.Preferably, the second vector is present in the medium in an amountwhich is about 1.75 to about 2.25 times as much as the amount of thefirst vector. More preferably, the second vector is present in themedium in an amount which is about twice as much as the amount of thefirst vector.

The invention further provides a second method of producing arecombinant protein. The second method comprises culturing cells, whichhave been contacted with a recombinant transient expression vectorencoding the protein, in a medium in a membrane-enhanced culturingvessel, whereupon a recombinant protein is produced. The second methodcan alternatively comprise culturing cells, which have been contactedwith a recombinant transient expression vector encoding the recombinantprotein, in a medium in a Fernbach flask, whereupon a recombinantprotein is produced. The recombinant transient expression vector can beany suitable such vector and preferably is an inventive recombinantexpression vector as described herein.

In a third method, the recombinant protein is produced upon contactingcells with at least one of the inventive recombinant expression vectorsdescribed herein. In a fourth method, the recombinant protein isproduced upon culturing any of the inventive host cells comprising anyof the inventive recombinant expression vectors described herein.

Any suitable method can be employed to contact cells with a firstvector, a second vector, or a recombinant expression vector, such thatthe cells express the protein encoded by the vector. Methods ofcontacting cells, such that the cells are modified to express aparticular protein, polypeptide, or peptide, are well-known in the art.See the references listed in Sambrook et al. (1989), supra. Suitablemethods of contacting cells to this end include, for instance, infectionwith a viral vector, transfection with a lipofection reagent, cationicpolymer, DEAE, or calcium phosphate, and electroporation.

The cells can be contacted with a first vector, a second vector, or arecombinant expression vector in the presence of a suitable cationicpolymer. Suitable cationic polymers for transfecting cells are known inthe art, and include, for example, polylysine and polyethyleneimine(PEI). In a preferred embodiment of the inventive method, the cationicpolymer is PEI. PEI can be linear or branched and can vary in molecularweight, depending on the number of base units, which comprise thepolymer. Preferably, the PEI is a linear PEI. More preferably, thelinear PEI has a molecular weight of about 25 kDa. Although the amountof PEI used in the method can be any amount, it is preferred that thelinear PEI is present in an amount that is about 1.5 to about 4.5, e.g.,1.5, 1.6, 1.75, 2.0, 2.25, 2.5, 2.6, 2.7, 2.75, 2.8, 2.9, 3.0, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.75, 3.8, 3.9, 4.0, 4.1, 4.25, 4.3, 4.4, and 4.5,times the amount of the vector(s) contacting the cells. Preferably, thePEI is present in an amount that is about 2.5 to 3.5 times the amount ofthe vector(s) contacting the cells. More preferably, the PEI is presentin an amount that is about twice the amount of the vector(s) contactingthe cells.

For purposes of the inventive method comprising contacting cells withmore than one vector, e.g., a first vector and a second vector, thecells can be contacted with the first vector and second vector in asequential fashion, e.g., first vector contacted with the cells beforethe second vector. Alternatively, the cells can be contacted with thefirst vector and second vector simultaneously. Preferably, the cells arecontacted with the first vector and second vector simultaneously. Forexample, in a method comprising contacting cells with more than onevector, the cells can be contacted with a first vector before orsimultaneously with a second or additional vector.

As used herein, the term “culturing” is synonymous with “maintaining.”Methods of culturing cells are known in the art (see, e.g., TissueEngineering Methods and Protocols, Morgan and Yarmush (eds.), HumanaPress, Inc., Totowa, N.J., 1999). As one ordinarily skilled recognizes,the conditions under which cells are cultured varies depending on thecell type. The conditions include temperature of the environment, theculturing vessel containing the cells, the composition of the variousgases, e.g., CO₂, which comprises the cell culture atmosphere orenvironment, the medium in which the cells are maintained, thecomponents and pH of the medium, the density at which cells aremaintained, the schedule by which the medium needs to be replaced withnew medium, etc. These parameters are often known in the art or can beempirically determined. For example, with respect to the inventivemethods, wherein cells are cultured in a medium, e.g., a first medium, asecond medium, etc., any method can be employed to culture the cells inthe medium, such that the cells express (and, in some instances,secrete) the protein encoded by the vector, which was contacted to thecells.

The cells are desirably cultured in a membrane-enhanced culturing vesselor a Fernbach flask. For purposes herein, the term “membrane-enhancedculturing vessel” refers to a container for holding cell cultures thathave been improved upon by the addition of at least one membrane.Suitable membrane-enhanced culturing vessels include membrane-based cellculture vessels, dialysis-based cell culture vessels, membrane-basedhigh density cell culture vessels, and two-compartment vessels. The term“vessel” as used herein is synonymous with systems, reactors,bioreactors, flasks, and devices. Suitable membrane-enhanced culturingvessels include, for instance, miniPerm® flasks, OptiCell® flasks, andthe CELLINE™ CL 1000 (referred to herein as Integra flasks or IntegraCL1000 flasks), which are commercially available from companies, such asIBS Integra Biosciences AG (Chur, Switzerland), OptiCell (Westerville,Ohio), VWR, Fisher Scientific, and Labmate (Asia). Most preferably, themembrane-enhanced culturing vessel is an Integra CL1000. For example,one of the ordinary skill in the art appreciates that amembrane-enhanced culturing vessel such as an Integra flask may comprisea nutrient chamber and a cultivation chamber, wherein nutrients from amedia reservoir in the nutrient chamber pass through a semi-permeablemembrane into the cultivation chamber containing cells so as to providea continuous supply of nutrients and wherein the membrane also allowsfor diffusion of metabolites out of the cultivation chamber and awayfrom contact with the cells but does not permit diffusion of arecombinant protein produced by the cells (e.g., an antibody or antibodyfragment) out of the cultivation chamber, and further wherein the cellsalso have sufficient gas exchange such as access to oxygen and carbondioxide through a separate silicone membrane at the bottom of thevessel.

As used herein, the term “Fernbach flask” refers to a commerciallyavailable Corning® polycarbonate Erlenmeyer flask having the Fernbachdesign. Such flasks are commercially-available from companies such asLife Sciences.

Without being bound to any particular theory, membrane-enhanced flasks(e.g., Integra CL1000, OptiCell® flasks, and miniPerm® flasks) andFernbach flasks are particularly suitable for culturing transfectedcells, for example, transiently transfected cells, as these devicespermit efficient gas exchange between the cells and the environment,e.g., the incubator environment, which permits optimal cell growth andproduction of the recombinant protein. Under certain conditions, shakeflasks can also be suitable culturing vessels in which cells can becultured for optimal cell growth and production of the recombinantprotein. It should be understood that any flask or culturing vessel thatpermits efficient gas exchange between the cells and the environment areincluded in the scope of the invention and are not limited to only theaforementioned flasks and culturing vessels.

In the inventive methods comprising culturing cells, the medium can beany suitable medium for culturing cells known in the art. The medium canbe, for example, a culture medium containing 1% low immunoglobulin (Ig)fetal bovine serum (FBS). Alternatively, the medium can be a serum-freecell culture medium, e.g., IS293™ medium. In some instances, the mediumis preferably a serum-free IS293™ medium (Irvine Scientific, Irvine,Calif.).

The cell cultures of the inventive methods can be initiated or seeded atany suitable cell density. As one of ordinary skill in the artrecognizes, the seeding density depends on a variety of factors, such ascell type, culturing conditions, and the day which has been selected forharvesting or purifying the recombinant protein from the cell culture.Desirably, the cell density is within the range of about 1.0×10⁶ toabout 2.0×10⁷ (e.g., about 1.0×10⁶ to about 1.5×10⁷). More preferably,the initiating seeding cell density of the cell culture is about 3.0×10⁶to about 1.0×10⁷. Without being bound to any particular theory, it isbelieved that the seeding density of cells, which have been transientlytransfected with a vector encoding a protein, is a factor in obtainingefficient production of a recombinant protein.

For purposes of the inventive methods, the cells that are cultured orare contacted with a first vector, a second vector, or a recombinantexpression vector can be any cell, such as those described herein as“host cells.” For example, the cells that are cultured and/or contactedwith one or more than one recombinant expression vector can be any hostcells. Preferably, the cells are mammalian cells, and, more preferably,the cells are human cells. The cells are desirably human embryonickidney cells. In a most preferred embodiment, the human embryonic kidneycells express Epstein-Barr virus nuclear antigen-1 protein (EBNA-1),e.g., 293E cells.

Cells, which have been contacted with a recombinant transient expressionvector, can be obtained by transiently transfecting cells by any methodknown in the art, including those described herein. Recombinanttransient expression vectors are known in the art and include, forinstance, pCEP4, pcDNA3, and any of the recombinant expression vectorsdescribed herein which comprise an oriP. Preferably, the recombinanttransient expression vectors are pMXT vectors. For example, the vectorscan be any of the inventive recombinant expression vectors as describedherein.

With respect to the first method of producing a recombinant protein(e.g., comprising contacting cells with a first vector and a secondvector), the method can further comprise the second inventive method ofproducing a recombinant protein. That is, the method of producing arecombinant protein can further comprise the step of culturing thecells, which have been contacted with a first vector and a secondvector, in a second medium in a membrane-enhanced culturing vessel(e.g., an Integra CL1000, an OptiCell® flask, a miniPerm® flask), aFernbach flask, or like flask. In such an embodiment, the second mediumcan be different from the medium in which the first and second vectorsare present. For purposes of the methods, which comprise culturing cellsin a membrane-enhanced culturing vessel, a Fernbach flask, or likeflask, the suitable medium for use in such a vessel or flask can be aserum-free cell culture medium, e.g., IS293 medium. Preferably, themedium is serum-free IS293 medium (Irvine Scientific, Irvine, Calif.).

With respect to the second inventive method of producing a recombinantprotein, the method can comprise the first inventive method of producinga recombinant protein. One of ordinary skill in the art recognizes thatthe methods described herein can be combined in such a way, such thatall of the limitations of the methods are met. Such a combined method iswithin the scope of the invention.

With respect to any of the inventive methods comprising culturing cells,e.g., in a membrane-enhanced culturing vessel, a Fernbach flask, or likeflask, the method can further comprise purifying or isolating therecombinant protein from the medium, e.g., the serum-free medium. Asused herein, the terms “purifying” and “isolating” do not necessarilyrefer to absolute purity or isolation, as one of ordinary skill in theart appreciates that a partially purified or partially isolated proteincan be useful or of value.

Methods of purifying proteins from mixtures are known in the art.Suitable purification methods include, for example, chromatography,electrophoresis, and the like. Suitable chromatographic methods ofpurifying polypeptides include, for example, HPLC, ion-exchangechromatography, affinity chromatography, etc. Preferably, the purifyingcomprises chromatographing the medium through a resin, such as acationic resin, an anionic resin, and an affinity resin. If thepolypeptide is an immunoglobulin chain, the purifying preferablycomprises the use of resin comprising Staphylococcus aureus Protein A,which is a bacterially-produced protein that binds to the Fc regions ofIgG antibodies. More preferably, the purifying comprises centrifugingthe medium through a column comprising Protein A, e.g., centrifuging themedium through a Protein A spin column (which is commercially availablefrom Pro-Chem).

The purifying can occur at any point in time after culturing the cells,which have been contacted with a vector. In some instances, it ispreferable for the purifying to occur after about 3 days of culturing,e.g., after about 3, 4, 5, 6 or more days. In other instances, it ispreferable for the purifying to occur after about 7 days of culturing,e.g., after about 7, 8, 9, 10, 11, 12, 13, 14, 15 or more days.

The invention provides fast and efficient methods of producing highlevels of recombinant proteins. In some instances, at least 300 μg/mlrecombinant protein is produced after 3 days of culturing. In otherinstances, at least 500 μg/ml recombinant protein is produced after 3days of culturing. In some preferred instances, at least 700 μg/mlrecombinant protein is produced after 3 days of culturing.

The term “recombinant protein” as used herein, refers to any protein orpart thereof that is produced by a genetically-engineered organism. Forexample, the recombinant protein can be any of the proteins describedherein.

For purposes of the first method of producing a recombinant protein, therecombinant protein is a heterodimeric protein or a heteromultermericprotein, such as a tetramer, which comprises two copies of two differentpolypeptide chains. Such proteins are known in the art, and include, forinstance, hemoglobin, immunoglobulins, T cell receptors, and B cellreceptors, etc. In a preferred embodiment of the first inventive method,the recombinant protein is a heterotetrameric protein. Desirably, theheterotetrameric protein is an immunoglobulin. In this instance, it ispreferred that the first vector encodes a heavy chain of animmunoglobulin, or a part thereof, and the second vector encodes a lightchain of an immunoglobulin, or a part thereof. The heavy chain can beany heavy chain of any immunoglobulin, as described herein. The lightchain can be any light chain of any immunoglobulin, as described herein.Exemplary antibody heavy and light chains: an LDP-01 heavy chain, whichis encoded by the nucleotide sequence of SEQ ID NO: 14 and comprises theamino acid sequence of SEQ ID NO: 15, and an LDP-01 light chain, whichis encoded by the nucleotide sequence of SEQ ID NO: 16 and comprises theamino acid sequence of SEQ ID NO: 17. The LDP-01 antibody is referred toherein as Ab#1 and has been described in WO 2004/033693(PCT/US2003/010154) and U.S. Patent Application Publication No.2003/0203447 A1.

EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the construction of recombinant expressionvectors of the invention.

Transient expression vectors for expression of any gene were constructedwith a multilinker site containing unique restriction sites positionedbetween the 3′ end of the CMV promoter and the 5′ end of the mouse lightchain 3′ untranslated region. Transient expression vectors containingcDNAs, which encode light chain κ or λ genes or heavy chain γ₁, γ₂, orγ₄ genes, under the control of a CMV promoter (Boshart et al., Cell 41:521-530 (1985)) and mouse light chain 3′ untranslated region (Xu et al.,J. Biol. Chem. 261: 3838-3845 (1986)) were contructed. Uniquerestriction sites were positioned at the 5′ end of the V region (e.g.,SalI) and in the junction regions between the V and constant regions(BlpI for heavy chain, BsiWI for K light chain and AvrII for lambda) forthe cloning of any new V region adjacent to the desired cognate constantregion. The vectors also contained the Epstein Barr virus oriP sequence(Reisman et al., Mol. Cell. Biol. 5: 1822-1832 (1985)) for episomalplasmid replication in 293E cells, the origin of replication from thevector pUC19, and the gene encoding resistance to ampicillin forselection of tranformants in E. coli. The transient expression vectorscontaining the multilinker sites, the heavy chain, and the light chainare shown in FIGS. 1A-1C.

Example 2

This example demonstrates a method of transiently transfecting cells forproducing recombinant proteins.

293E cells (Invitrogen, R620-07) were maintained as adherent cultures inDulbecco's Modified Eagle Medium (DMEM) (Gibco-Invitrogen) supplementedwith 10% fetal bovine serum (FBS, Hyclone), 2 mM glutamine, and 250μg/ml G418 antibiotic (Gibco-Invitrogen). For growth in suspensionculture, the cells were adapted to the following serum-free mediaformulations: IS293™ (Irvine Scientific), IS293-V™ (Irvine Scientific),293 SFM II (Gibco-Invitrogen), H-SFM (Gibco-Invitrogen), and HYQ®PF293(HyClone). The cells were originally supplemented with 10% low IgG FBS(HyClone) and 2 mM glutamine and gradually weaned down to 1% low IgG FBSover a period of several weeks. Once in 1% low IgG FBS, the cells weretransferred to shake flasks for continued adaptation to suspensiongrowth. Growth and viability were monitored using the VICELL™ XR CellViability Analyzer (Beckman-Coulter).

All plasmids were transformed into DH5α cells (Invitrogen) and purifiedusing endotoxin-free plasmid purification kits (QIAGEN®). Fortransfections in 6-well plates, 2 ml of cells at 5×10⁵ cells/ml wereseeded per well. For transfections in shake flask cultures, cells wereseeded at 8×10⁵ cells/ml at the appropriate volumes prior totransfection. DNA (2 μg/ml) was pre-incubated with linearpolyethyleneimine (PEI, 25 kDa MW, Polysciences) at a concentration of 4μg/ml for 10 min at room temperature prior to addition to cells. TheDNA/PEI mixture was then added to cells, and the cells with the DNA/PEIwere either maintained in shake flasks or transferred to Integra flasks.

Example 3

This example demonstrates the determination of the optimal PEI:DNA ratiofor transient transfections.

Adherent 293E cells grown in DMEM supplemented with 10% FBS in 6-wellplates were transfected with pQBI-pGK (GFP expressing plasmid,Q-biogene) using linear polyethyleneimine (PEI) as described in Example2. DNA (1 μg/ml, 2 μg/ml, or 5 μg/ml) was pre-incubated with linear PEI(1, 2, 4, 5, 10, or 25 μg/ml) for 10 min at room temperature prior tothe addition to cells, then the PEI/DNA mixture was added to cells, andthe cells were maintained in shake flasks or Integra flasks.

GFP expression was monitored 24 hours post-transfection using a BectonDickinson FACScan flow cytometer equipped with the Cytek AutomatedMicrosampler System (AMS) 96-well plate reader. Flow data was analyzedusing FlowJo (Tree Star, Inc.). Cells also were counterstained with 1μg/ml propidium iodide (PI) to determine cell viability. Growth andviability of the cells post-transfection were monitored using theVICELL™ XR Cell Viability Analyzer (Beckman-Coulter). The cellstransfected with 1 μg/ml DNA and 1 μg/ml PEI (FIG. 2A); 2 μg/ml DNA and2 μg/ml PEI (FIG. 2B); 5 μg/ml DNA and 5 μg/ml PEI (FIG. 2C); 1 μg/mlDNA and 2 μg/ml PEI (FIG. 2D); 2 μg/ml DNA and 4 μg/ml PEI (FIG. 2E); 5μg/ml DNA and 10 μg/ml PEI (FIG. 2F); 1 μg/ml DNA and 5 μg/ml PEI (FIG.2G); 2 μg/ml DNA and 10 μg/ml PEI (FIG. 2H); and 5 μg/ml DNA and 25μg/ml PEI (FIG. 2I) were measured for GFP expression (x-axis) and PIstaining (y-axis) by flow cytometry, and the resulting data was plottedin the series of graphs of FIGS. 2A-2I.

As shown in FIGS. 2A-2I, the DNA concentration of 1 μg/ml at a PEI:DNAratio of 2:1 gave the highest percentage of cells expressing GFP withrelatively low cellular cytotoxicity 24 hours post-transfection.

The results of this example demonstrated the production of recombinantprotein and confirmed that the optimal PEI:DNA ratio for transienttransfection is 2:1.

Example 4

This example demonstrates the determination of the optimal medium forculturing transiently transfected cells.

293E cells were grown and transfected in the presence of 1% low-IgGserum in 6-well plates and shake flasks, as described in Example 2.Twenty-four hours after transfection, 293E cells were adapted tosuspension growth in one of 5 different serum-free media formulations(IS293™, H—SFM, IS293V™, SFMII, or HYQ®PF293) or one serum-containingmedia formulation (DMEM) as in Example 2.

Twenty-four to forty-eight hours later, GFP expression by transfectedcells were determined as described in Example 3. Cells also werecounterstained with 1 μg/ml PI to determine cell viability. Growth andviability of the cells post-transfection were monitored using theVICELL™ XR Cell Viability Analyzer (Beckman-Coulter). The resulting datafrom GFP expression (bars) and for PI staining (X) was plotted to formthe graph of FIG. 3.

As shown in FIG. 3, IS293™ medium (Irvine Scientific) gave the highestpercentage of GFP-expressing cells with minimal cytotoxicity in shakeflasks; values were comparable to those obtained with adherent 293Ecells cultured in DMEM supplemented with 10% FBS.

The results of this example demonstrated that IS293™ medium is theoptimal serum-free medium to be used with transiently transfected cellsfor producing recombinant proteins.

Example 5

This example demonstrates the determination of optimum heavy and lightchain plasmid ratios for maximum antibody productivity.

Various ratios of pMXT (heavy chain (HC)):pMXT (light chain (LC)) (seeExample 1) or pCEP4 (HC):pCEP4 (LC) were tested for effects on antibodyproductivity in cells grown in IS293 medium supplemented with 1% low-IgGserum in shake flasks. The pCEP4 vector containing the nucleotidesequence encoding the Ab#1 heavy chain (SEQ ID NO: 14) was constructedby cloning the coding sequence into KpnI and Xho sites. The pCEP4 vectorcontaining the nucleotide sequence encoding the Ab#1 light chain (SEQ IDNO: 16) was constructed by cloning the coding sequences into Nhe and Xhosites. The encoded heavy chain and light chain of Ab#1 is set forth asSEQ ID NOs: 15 and 17, respectively. All plasmids were amplified bytransformation into DH5 α cells and purified as described in Example 2.293E cells were transiently transfected as described in Example 2.Transfected cells were transferred to IS293™ medium in shake flasks for7-10 days. Antibody expression by the cells transfected with a 1:1, 1:2,or 2:1 ratio of vector encoding HC:vector encoding LC, wherein thevectors were either pMXT or pCEP4 was determined by sandwich ELISA andthe data analyzed in PRISM™ (GraphPad). The resulting data was plottedto form the graph of FIG. 4.

As shown in FIG. 4, a 1:2 ratio of HC:LC generated the highest antibodyproductivity with Ab#1 achieving levels of 60-70 μg/ml after 7-10 days.The highest productivity for Ab#1 (LDP-01) in pMXT was ˜3× greater thanthe best output achieved using pCEP4.

The results of this example demonstrated that the pMXT vector is optimalfor co-transfecting cells with vectors encoding different polypeptidechains at a ratio of 1:2.

Example 6

This example demonstrates that the level of antibody production bytransiently transfected cells cultured post-transfection inmembrane-enhanced culturing vessels are comparable to the level ofantibody production achieved by transfected cells culturedpost-transfection in shake flasks.

293E cells were transiently transfected in shake flasks as described inExample 2. Cells were either maintained in the shake flasks ortransferred to 15 ml of medium and placed in Integra CL1000 flasks.After 7-10 days, cell culture supernatant was harvested, clarified, andpurified for antibodies using a standard Protein A column, if cells werecultured in shake flasks, or a Protein A spin column, if cells werecultured in Integra flasks. Cell viability and antibody production ofboth sets of cells 0, 4, 7, and 14 days post-transfection were assayedas described in Examples 4 and 5, respectively. For antibody expressionusing the Integra CL1000 flask, 200 ml of transfected 293E cells wereresuspended in 15 ml of IS293™ medium supplemented with 1% low IgG FBSand 250 μg/ml G418 antibiotic and transferred into the membranecompartment. One liter of IS293™ medium was added to the upper mediachamber.

The cell viability (X) and antibody production (•) of the transfectedcells maintained in Integra flasks are shown in FIG. 5A, whereas thelevels of antibody production for Ab#1 by cells maintained in eithershake flasks or by Integra CL 1000 flasks are shown in FIG. 5B.

As shown in FIG. 5A, antibody production of cells cultured in Integraflasks peaked at 7 days, producing over 1 mg/ml antibody. This level iscomparable to the level of antibody production of transientlytransfected cells cultured in shake flasks as shown in FIG. 5B.

The results of this example demonstrated that Integra flasks aresuitable culturing vessels for maintaining small volumes of transientlytransfected cells. The small volume permits the use of Protein A spincolumns, which facilitates the purification of antibodies from the cellculture supernatant.

Example 7

This example demonstrates a method of producing antibodies inmembrane-enhanced culturing vessels at optimized seeding densities.

Suspension-adapted HEK 293E cells were maintained in IS293™ medium(Irvine Scientific) supplemented with 1% low IgG FBS (HyClone), 2 mMglutamine (Gibco-Invitrogen), and 250 μg/ml G418 antibiotic(Gibco-Invitrogen). For transfection, cells were seeded at 8×10⁵cells/ml in shake flasks at the appropriate volumes prior totransfection. DNA encoding Ab#1 or Ab#2 (which differed from Ab#1) waspre-incubated with linear polyethyleneimine (PEI, 25 kDa MW,Polysciences) at optimized conditions (see, e.g., Example 3; see also,e.g., Handa et al., American Society for Cell Biology, posterpresentation #1937 (2004)) prior to addition to cells. For antibodyexpression using the Integra CL1000 flask, cells at the followingseeding densities were resuspended in 30 ml of IS293™ mediumsupplemented with 1% low IgG FBS, 2 mM glutamine, and 250 μg/ml G418antibiotic and transferred into the cultivation chamber: 1.3×10⁶ (I-50),2.7×10⁶ (I-100), 5.3×10⁶ (I-200), and 1.1×10⁷ (I-400). For comparison,8×10⁵ cells (E-200) were seeded in Erlenmeyer flasks. All flasks wereincubated for 3, 5, 7, or 10 days post-transfection. One ml samples fromthe nutrient chambers and cultivation chambers of the Integra CL 1000flasks were removed and analyzed at 3, 5, 7, or 10 dayspost-transfection.

Growth and viability were monitored using the VICELL™ XR Cell ViabilityAnalyzer (Beckman-Coulter). The percentage of viable cells 1, 3, 5, 7,and 10 days after transfection for cells tranfected at different seedingdensities is shown in FIGS. 6A and 6B. The viable cell count of cells 0,1, 3, 5, 7, and 10 days after transfection for cells transfected atdifferent seeding densities is shown in FIGS. 7A and 7B.

As shown in FIGS. 6A (Ab#1), 6B (Ab#2), 7A (Ab#1) and 7B (Ab#2), cellviability did not vary between flasks, but viable cell growth wasimproved in the Integra flasks for all seeding densities tested. Maximumdensities of 3-5×10⁷ cells/ml were achieved for all conditions over the10 day analysis period.

Analytes, gases, and pH of the samples were determined 3, 5, 7, and 10days post-transfection using a BIOPROFILE™ Chemistry Analyzer (NovaBiomedical). The data for selected nutrients and metabolites of themedia containing cells producing Ab#1 or the media containing no cells(Media Only) are set forth in Table 1.

TABLE 1 Nutrient Sample Flask Chamber Media Only Day 3 Day 5 Day 7 Day10 Glucose I-50 cultivation 5.37 3.35 2.85 2.57 2.76 (g/L) I-50 nutrient5.36 5.11 4.49 4.12 3.47 I-100 cultivation 5.37 3.86 2.93 2.99 2.41I-100 nutrient 5.36 5.05 4.39 3.97 3.85 I-200 cultivation 5.37 3.27 3.093.01 2.84 I-200 nutrient 5.36 4.62 4.12 3.68 3.10 I-400 cultivation 5.372.86 3.11 2.98 2.74 I-400 nutrient 5.36 4.52 3.94 3.67 3.23 E-200 N/A5.36 4.42 2.51 2.36 2.06 Glutamine I-50 cultivation 6.96 6.07 4.77 4.804.32 (mmol/L) I-50 nutrient 6.82 6.30 5.89 5.54 4.79 I-100 cultivation6.96 5.84 4.57 4.65 4.03 I-100 nutrient 6.82 6.37 5.88 5.41 5.10 I-200cultivation 6.96 5.36 4.64 4.50 4.68 I-200 nutrient 6.82 6.07 5.62 5.194.63 I-400 cultivation 6.96 5.47 5.25 5.02 4.21 I-400 nutrient 6.82 6.375.91 5.68 5.12 E-200 N/A 6.82 6.31 5.82 5.53 5.08 Lactate I-50cultivation 0.26 1.92 2.42 3.17 2.82 (g/L) I-50 nutrient 0.34 0.74 1.361.82 2.27 I-100 cultivation 0.26 1.85 2.43 2.74 2.82 I-100 nutrient 0.340.99 1.62 1.94 2.52 I-200 cultivation 0.26 2.47 2.32 2.84 2.72 I-200nutrient 0.34 1.25 1.86 2.09 2.33 I-400 cultivation 0.26 2.57 2.31 2.782.66 I-400 nutrient 0.34 1.63 2.17 2.35 2.38 E-200 N/A 0.34 1.53 2.502.59 2.48

As shown by the resulting data, transiently transfected cells maintainedin 30 ml media in the cultivation chamber of an Integra CL 1000 flaskcan reach cell densities of up to 3-5×10⁷ viable cells/ml (e.g.,4.5×10⁷). Nutrients from the media reservoir in the nutrient chamberpass through a semi-permeable membrane into the cultivation chamberproviding a continuous supply of essential nutrients. The membrane alsoallows for diffusion of metabolites out of the cultivation chamber andaway from contact with cells. Cells also have efficient access to oxygenand carbon dioxide through a separate silicone membrane at the bottom ofthe flask.

The Integra supernatant from the cultivation chamber had higher glucoselevels than shake flasks but lower glutamine levels. The levels oflactate appeared similar between the two cultures. The higher relativelevels of glucose to lactate in the Integra cultures could indicate thatthe cells are generating more ATP by promoting efficient entry ofpyruvate from glycolysis into the TCA cycle.

Antibody titers of transfected cells placed in Integra flasks or shakeflasks at different seeding densities were determined using theEASY-TITER™ Human IgG Assay Kit (Pierce) 0, 3, 5, 7, and 10 dayspost-tranfection. The data expressed as the concentration of antibodytiters (μg/ml) is shown in FIGS. 8A (Ab#1) and 8B (Ab#2), whereas thedata expressed as the total antibody yield (mg) is shown in Table 2.

TABLE 2 Day 3 Day 5 Day 7 Day 10 % of E-200 % of E-200 % of E-200 % ofE-200 Sample Yield (mg) Max Yield Yield (mg) Max Yield Yield (mg) MaxYield Yield (mg) Max Yield Ab#1 E-200 7.6 52% 14.5 100% 7.4 51% 7.6 52%I-50 0.8 6% 5.2 36% 8.5 59% 13.3 92% I-100 5.2 36% 8.3 57% 14.7 101%24.0 166% I-200 9.4 65% 13.8 95% 13.8 95% 19.0 131% I-400 11.00 76% 15.7108% 14.3 99% 11.7 81% Ab#2 E-200 4.1 50% 6.3 77% 7.5 92% 8.2 100% I-501.2 15% 4.3 52% 7.6 93% 6.4 78% I-100 3.5 43% 8.3 101% 10.4 127% 12.9157% I-200 6.1 74% 13.0 159% 17.0 207% 22.0 268% I-400 10.8 132% 17.8217% 21.0 256% 22.0 268%

As shown in FIGS. 8A and 8B, antibody productivity for the twoantibodies tested, Ab#1 and Ab#2, were different in Erlenmeyer flasks.Ab#1 peaked early at day 5 (˜70 μg/ml), followed by a decrease inantibody concentration. Ab#2 showed slower and steadier productivityover the full 10 days, achieving a maximal antibody output of ˜40 μg/ml.For both Ab#1 and Ab#2, antibody productivity in the Integra flasksachieved steady levels of increasing Ab productivity over the 10 dayperiod. An exception was the I-400 sample for Ab#1, which showed slightdecreases in productivity at days 7 and 10; however, the decrease wassubstantially less as compared to that in the E-200 samples.

As shown in Table 2, the maximal yield for Ab#1 in the E-200 culture was˜15 mg at day 5. Comparable levels (≧90% of E-200 maximum) were obtainedin the I-200 and I-400 cultures at day 5 as well, and higher totalyields were obtained with I-100 and I-200 after day 10, namely 166% (24mg) and 131% (19 mg), respectively.

As also shown in Table 2, the maximal yield for Ab#2 in the E-200culture was ˜8 mg at day 10. Comparable levels (≧90% of E-200 maximum)were obtained as early as day 3 with I-400 (˜11 mg), at day 5 with I-100and I-200 (8 mg and 13 mg, respectively), and day 7 with I-50 (˜8 mg).Higher yields were obtained for I-100, I-200, and I-400 at day 10, 157%(13 mg), 268% (22 mg), and 268% (22 mg), respectively.

The results of this example demonstrated high levels of antibodyproduction in Integra flasks within short periods of time. Celldensities of 1.0×10⁶ and 1.5×10⁷ were examples of optimal densities forproducing high levels of antibodies. As shown herein, transientlytransfected cells, for example, 293E cells, in membrane-enhancingculturing vessels such as Integra flasks, generated higher totalantibody yields over cells cultured in Erlenmeyer flasks, irrespectiveof antibody productivity levels in shake cultures. Transientlyexpressing antibodies in membrane-enhancing culturing vessels such asIntegra flasks also appeared to better retain antibody stability uponexhaustion of the culture.

As shown by the results obtained herein, total antibody yields fromtransiently transfected 293E cells are significantly increased whencultured in Integra flasks vs. standard Erlenmeyer flasks. Increasingthe number of transfected cells seeded in the Integra flask cansubstantially decrease the time to reach maximum antibody yield, whiledecreasing the seeding density allows for multi-mg production ofantibodies using a fraction of the cells under normal conditions in anErlenmeyer flask. Generating transiently expressed antibodies in Integraflasks also better maintains the antibody titer for longer periods oftime thus allowing for greater confidence to allow cultures to proceedto extinction without significant loss of antibody. Advantageously,usage of membrane-enhancing culturing vessels, such as Integra flasks,for transient protein production, such as antibody production, allowsfor increased total yield, faster production by using more cells, and/orconservation of cells by using fewer cells while maintainingproductivity comparable to non-membrane culturing vessels, such asErlenmeyer flasks.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1: A recombinant expression vector comprising a promoter, a 3′ UTR of alight chain gene and an Epstein-Barr virus origin of replication (oriP).2: The recombinant expression vector of claim 1, wherein the light chaingene is a murine light chain gene. 3: The recombinant expression vectorof claim 2, wherein the 3′ UTR comprises a nucleotide sequence ofnucleotides 1062-2560 of SEQ ID NO:
 1. 4: The recombinant expressionvector of claim 1, wherein the recombinant expression vector is arecombinant transient expression vector. 5: The recombinant expressionvector of claim 1 further comprising a pUC19 origin of replication or a5′ UTR intron. 6: The recombinant expression vector of claim 5 furthercomprising a pUC19 origin of replication and a 5′ UTR intron. 7:(canceled) 8: The recombinant expression vector of claims 5 and 6,wherein the 5′ UTR intron comprises nucleotides 888-974 of SEQ ID NO: 1.9: The recombinant expression vector of claims 5 and 6, wherein thepUC19 origin of replication comprises the nucleotide sequence ofnucleotides 4551-5220 of SEQ ID NO:
 1. 10: The recombinant expressionvector of claim 1 further comprising an antibody signal sequence. 11:The recombinant expression vector of claim 1, wherein the recombinantexpression vector does not comprise an antibody signal sequence. 12: Therecombinant expression vector of claim 1 further comprising a nucleotidesequence encoding a protein or a functional fragment thereof. 13: Therecombinant expression vector of claim 12, wherein the nucleotidesequence encodes an immunoglobulin chain. 14: The recombinant expressionvector of claim 13, wherein the immunoglobulin chain is selected fromthe group consisting of a γ₁ heavy chain, a γ₂ heavy chain, a γ₄ heavychain, a κ light chain, and a λ light chain. 15: A method of producing arecombinant protein, comprising contacting a cell with the recombinantexpression vector of claim 1, whereupon a recombinant protein isproduced. 16: A host cell comprising the recombinant expression vectorof claim
 1. 17: The host cell of claim 16, wherein the host cell is amammalian cell. 18: The host cell of claim 17, wherein the mammaliancell is a human cell. 19: The host cell of claim 18, wherein the humancell is a human embryonic kidney cell. 20: The host cell of claim 19,wherein the human embryonic kidney cell expresses an Epstein Barr virusnuclear antigen-1 (EBNA-1) protein. 21: The host cell of claim 20wherein the host cell is a 293E cell. 22: A method of producing arecombinant protein, comprising culturing the host cell of claim 16,whereupon a recombinant protein is produced. 23-93. (canceled) 94: Therecombinant expression vector of claim 1, wherein the promoter is aviral promoter. 95: The recombinant expression vector of claim 94,wherein the viral promoter is a CMV promoter.